TECHNICAL ADVANCES: In collaboration with NHLBI investigator Gregory Kato and NIBIB investigator Alexander Gorbach, we have analyzed data from infrared imaging of the human forearm with respect to forearm blood flow measured by conventional strain gauge plethysmography. We observed strong correlations between blood flow and skin temperature at baseline, and in response to intra-arterial administration of endothelium-dependent and endothelium-independent vasodilators. This demonstrates that infrared imaging can be used to quantify blood flow non-invasively in human subjects. Furthermore we found that baseline skin temperature was predictive of the vasodilatory response to exogenous nitric oxide, illustrating the potential of skin temperature to serve as a physiologic biomarker of vascular function. In collaboration with Glenn Nardone at the NIAID RTB we have successfully developed an assay for the sensitive detection and quantification of Arginine, homoarginine, ADMA, and SDMA. This assay improves upon existing published techniques to achieve a greater level of sensitivity and accuracy. We are able to measure 0.3 uM concentrations in sample volumes of only 5 uL, this makes the technique not only amenable analysis of human plasma samples, but also useful for cell cultures and small animal models where sample volume may be limiting. The inter-day CV is between 1 and 6%, demonstrating excellent reproducibility. CLINICAL / BASIC SCIENCE ADVANCES: We have applied this HPLC technique to the analysis of arginine metabolites in two separate populations with malaria infection, Cambodian adults and Gambian children. In collaboration with Chanaki Amaratunga, Rick Fairhurst and Duong Socheat, we have analyzed arginine metabolites in Cambodian adults with mild (N = 75) and severe (N = 22) Plasmodium falciparum malaria infection and compared them against healthy Cambodian individuals (N = 34) of the same age and geographic region. Plasma arginine concentration was 123 uM (IQR 100-143) in healthy individuals, but fell to 72 uM (IQR 56-111) in patients with mild malaria, and 60 uM (IQR 41-97) in patients with severe malaria (p <0.0001 for healthy vs malaria-infected patients). Plasma ADMA concentration also decreased in malaria-infected patients compared to healthy individuals (Healthy 0.4 uM, IQR 0.34 - 0.41;Mild 0.34 uM, IQR 0.28 - 0.42;Severe 0.34 uM, IQR 0.30 - 0.52;p = 0.02 for healthy vs malaria-infected patients). Conversely, plasma SDMA increased in malaria-infected patients compared to healthy individuals (Healthy 0.36 uM, IQR 0.30 - 0.40;Mild 0.43 uM, IQR 0.35 - 0.54;Severe 0.49 uM, IQR 0.40 - 0.57;p = 0.0003 for healthy vs malaria-infected patients). The proportion of methylarginine to arginine was significantly elevated in the malaria-infected patients, and would lead to diminished nitric oxide production during malaria infection, a potential mechanism explaining the low nitric oxide bioavailability and endothelial cell dysfunction observed in malaria. In collaboration with Michael Walther at the LMIV and co-workers at the MRC Laboratories in The Gambia, we examined arginine and methylarginine concentration in plasma from Gambian children with mild and severe malaria. Similar to the Cambodian analysis, we found plasma arginine to be severely depleted in Gambian children with malaria (Healthy 94 uM, IQR 87-102;Mild 45 uM, IQR 35-56;Severe 32 uM, IQR 23-41;p <0.001 for all comparisons). ADMA was also significantly lower in malaria-infected compared to healthy individuals (Healthy 0.61 uM, IQR 0.58-0.71;Mild 0.40 uM, IQR 0.33-0.47;Severe 0.40 uM, IQR 0.30-0.51;p <0.001 for healthy vs malaria patients). Contrary to the observation in Cambodian adults, Gambian children had diminished SDMA levels during malaria infection (Healthy 0.48 uM, IQR 0.45-0.51;Mild 0.39 uM, IQR 0.31-0.50;Severe 0.43 uM, IQR 0.29-0.54;p <0.001 for healthy vs mild patients, p <0 .05 for healthy vs severe). We were surprised to find that plasma methylarginine concentration was lower in malaria patients compared to healthy Gambian children. One potential mechanism is that the profound arginine depletion limits the amount of arginine available for methylation by PRMT. To test this hypothesis, we analyzed the correlations between arginine and ADMA, and arginine and SDMA. Interestingly, we found methylarginine concentrations to be highly dependent on arginine concentrations during acute malaria infection, but not in healthy children or children recovered from malaria. For ADMA, the correlation between ADMA and arginine was modest in healthy children (r = 0.43, p = 0.02), and was stronger in mild malaria patients (r = 0.59, p = 3e-08) and strongest in severe malaria patients (r = 0.77, p = 1e-16). In children recovered from malaria the correlation returned to normal at r = 0.39 and r = 0.49. For SDMA the change in arginine dependence was even more pronounced. There was no correlation between SDMA and arginine in healthy children (r = 0.04, p = 0.85), but there was a strong correlation in patients with mild malaria (r = 0.42, p = 1e-04), and strongest in patients with severe malaria (r = 0.59, p = 6e-09). There was weak or no correlation between SDMA in children recovered from mild and severe malaria (r = 0.30, p = 0.02 and r = 0.20, p = 0.17). These data together show that during acute malaria infection arginine methylation is limited by the availability of arginine. This may be an important homeostatic mechanism for the regulation of nitric oxide synthase activity: as the concentration of arginine (the substrate for nitric oxide synthase) falls, the concentration of ADMA and SDMA (nitric oxide synthase inhibitors) will fall as well -- acting to maintain NOS activity in the setting of arginine depletion. However, the data reveal that during an acute malaria infection, homeostasis was not maintained. While the concentration of ADMA and SDMA became highly arginine-dependent during malaria infection, the proportion of methylated arginine increased. In healthy Gambian children, ADMA was 0.7% (IQR 0.6 - 0.8%) the concentration of arginine. In patients with mild malaria, ADMA increased to 0.9% (IQR 0.7 - 1.2%, p = 7e-05) and in patients with severe malaria ADMA increased to 1.4% (IQR 1.1 - 1.7%, p = 8e-14), representing a doubling of the ratio of ADMA:Arginine. In patients recovered from severe malaria, ADMA returned to 0.7% (IQR 0.6 - 1.0%, p = 0.25 compared to healthy children). A similar effect was seen for SDMA. In healthy Gambian children, SDMA was 0.5% (IQR 0.5 - 0.6%) the concentration of arginine. In patients with mild malaria, SDMA increased to 0.9% (IQR 0.7 - 1.1%, p = 4e-10), and in patients with severe malaria, SDMA increased to 1.4% (IQR 1.1 - 1.8, p = 3e-15) a doubling of the ratio of SDMA:Arginine. In patients recovered from severe malaria, SDMA returned to 0.5% (IQR 0.4 - 0.7%, p = 0.99 compared to healthy children). In conclusion, the low nitric oxide bioavailability observed in acute severe malaria may be attributable to upregulation of arginine methylation or accelerated release of methylarginines from red blood cells, the major reservoir of methylarginine in blood, via known mechanisms such as proteasome activity or autophagy. This represents a novel mechanism of disease whereby infection alters nitric oxide synthase activity by increasing the relative concentration of methylated arginines. Both the physiologic impact of elevated ADMA:arginine and the relative contributions of host metabolic pathways versus parasite metabolic pathways remain to be elucidated in patients with severe malaria.
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